Method for enrichment of natural antisense messenger RNA

ABSTRACT

A method for enrichment of natural antisense mRNA which involves hybridization of cDNA obtained from sense RNA with cDNA obtained from antisense RNA, followed by DNA polymerase treatment of the sense-antisense hybrid DNA molecule. A natural antisense library can be generated by cloning of sense-antisense hybrid DNA molecules in a vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of applicationSer. No. 09/680,420, filed Oct. 6, 2000, which claims priority under 35U.S.C. §119(e) from U.S. provisional application No. 60/157,843, filedOct. 6, 1999, the entire contents of both applications being herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for enriching antisensemessenger RNA that are naturally expressed in cells.

[0004] 2. Description of the Related Art

[0005] The functional information of the genes in the genome isunidirectional. Taking as an example the most simple gene which encodesa protein, the RNAs encoding a protein are transcribed from one of thestrands and define one mRNA that possesses the information for thetranslation of a single protein. This simplistic view is obviously morecomplex for many genes due to mechanisms such as alternative splicingand intron encoded mRNAs. While this general view of the unidirectionalnature of genes is true with regard to structural information, it is notso with regard to regulatory information. The possibility oftranscription of the other strand of a gene, in the reverse direction,will result in the production of an RNA that is complementary to themRNA that possesses the structural information (the sense mRNA). SuchRNAs, termed antisense RNAs, have the potential to strongly bind to thesense mRNA, producing a double stranded RNA, and to inhibit therealization of the structural information. However, it is possible thatthese RNAs also possess structural information.

[0006] In general, natural antisense RNAs are endogenous transcriptscontaining regions complementary to transcripts of other genes or othertranscripts arising from the same gene locus. Antisense RNA is an RNAwhich contains a stretch of nucleotides complementary to another RNAthat has some cellular function. The length of the complementary stretchis usually a few hundred nucleotides, but shorter stretches can also beimportant. The expression of antisense RNA is a powerful way ofregulating the biological function of the sense RNA molecules. Naturalantisense RNAs have been shown to play important regulatory roles,including control of cell growth, malignant transformation and othercellular phenotypes. Through the formation of a stable duplex betweenthe sense RNA and antisense RNA, the normal or sense RNA transcript canbe rendered inactive and untranslatable.

[0007] Natural antisense transcripts can either be cis-encoded ortrans-encoded. Cis-encoded antisense arises from transcription of thecomplementary strand of the sense gene; both sense and antisensetranscripts originate from the same locus and thus, the antisensetranscripts have regions of perfect complementarity to the sense mRNA.Trans-encoded antisense arises from transcription of a different genomiclocus, and accordingly, it is expected that in such cases thecomplementarity of the antisense region will be not complete.

[0008] Following the discovery of natural antisense RNAs in prokaryotes,natural antisense RNAs were also discovered in a variety of eukaryotescovering a wide range of the phylogenetic tree, including viruses(Michael et al., 1994), slime molds (Lee et al., 1993), insects(Lankenau et al., 1994), amphibians (Kimelman et al., 1989), birds(Farrell et al., 1995) and mammals (Murphy et al., 1994). Moreover, mostof the genes for which endogenous antisense transcripts were discoveredencode proteins of key regulatory roles in important cellularphenotypes, such as cellular proliferation (Chang et al., 1991),apoptosis (Khochbin et al., 1989) and embryonic development (Bedford etal., 1995), or of key cellular processes such as translation (Noguchi etal., 1994), transcription (Krystal et al., 1990) and splicing (Fu etal., 1992). The thorough review article by Vanhee-Brossollet and Vaquero(1998) offer a summary of all antisense RNAs discovered so far and theirfunctional importance. The evidence suggests that control of geneexpression by endogenous antisense RNAs is one of the regulatorymechanisms in the cell and is widespread throughout the eukaryotickingdom.

[0009] All of the antisense transcripts discovered so far were found bysporadic experiment stemming from studies of single genes. This impliesthat the antisense regulatory mechanism might be of general importanceand relevant for many more genes. Thus, a general method for findingthose mRNAs in the cell for which an antisense RNA exists would be ofgreat value. The object of the approach and method of the presentinvention is to enable those in the art to investigate which mRNAs inthe cell have antisense RNAs, as compared to studies done up until nowwhich investigated the question “does this specific mRNA have anantisense RNA counterpart?”

[0010] Citation of any document herein is not intended as an admissionthat such document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

[0011] The present invention is directed to rapid methods for enrichmentof natural antisense mRNA (the exonuclease and polymerase activities ofDNA polymerases on the sense-antisense double-stranded hybrid), followedby amplification and cloning of its corresponding cDNA. Thus, theinvention overcomes the deficiencies in the prior art as discussedabove. These methods provide for the enrichment and detection of naturalantisense mRNA from any natural source of RNA. Poly A+ mRNA in a sampleof RNA is converted into single-stranded cDNA which is denatured todisrupt any secondary structure, and then allowed to re-anneal understringent conditions with any other cDNA having a segment with asignificant complementary sequence to form a hybrid molecule with adouble-stranded cDNA segment. Sense and antisense cDNAs hybridize toeach other and form double-stranded stretches or segments of DNA. In anembodiment of the present invention, the resulting hybridizationproducts with double-stranded DNA segments are treated with a DNApolymerase, such as T4 DNA polymerase, which has a 5′ to 3′ polymeraseactivity and a 3′ to 5′ exonuclease activity. The resultingdouble-stranded molecules are then amplified and cloned.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a schematic presentation of an embodiment of thenatural antisense enrichment procedure according to the presentinvention.

[0013]FIG. 2 shows an agarose gel electrophoresis analysis of PCRamplification products generated from single stranded cDNA obtained fromreverse transcription (RT) of c-erb, Ku autoantigen, and GAPDH beforeand after the antisense enrichment procedure according to FIG. 1.

[0014]FIG. 3 shows gene-specific RT-PCR analysis of the expression ofsense (S) and antisense (AS) mRNAs of clone N1-15 by use of specificprimers complementary to the two strands, and GAPDH control by agarosegel electrophoresis. Clone N1-15 (RbAp48 mRNA encoding retinoblastomabinding protein, GenBank accession number X74262) was obtained from theantisense enrichment procedure. The lane designated M is a lane ofmolecular weight markers.

[0015]FIG. 4 shows a Northern blot analysis of RbAp48 mRNAs with senseand antisense RbAp48 single stranded probes.

[0016]FIG. 5 shows a schematic presentation of a second embodiment ofthe natural antisense enrichment procedure according to the presentinvention.

[0017]FIG. 6 shows a schematic presentation of a variation of the secondembodiment shown in FIG. 5, where a 3′- end of the B-ssc DNA is blocked.

[0018]FIG. 7 shows a schematic presentation of hybrids generated thatare resistant to the antisense enrichment procedure according to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is based on the development of simple andrapid methods for the enrichment of natural antisense mRNA from anysource of mRNA. The source of the poly A+ mRNA used in the presentmethod can be a cell line, tissue, whole organism, or even a mixture ofpoly A+ mRNA from two or more sources. The source(s) of mRNA ispreferably a natural source(s). A mixture of RNA from different sourcescan be, for example, a mix of control mRNA derived from normal tissue orcells and mRNA from treated tissues or cells, or a mix of control mRNAand mRNA derived from pathologic tissue mRNA, etc.

[0020] A method for enrichment of natural antisense messenger RNA (mRNA)according to the present invention is schematically presented in FIG. 1.A population of single-stranded cDNA molecules is generated from asource of poly A⁺ RNA (RNA recovered from oligo dT columns after loadingthereon a sample of total RNA from cells or tissue), where such columnsbind the mRNA that contain a polyA stretch. The vast majority ofeukaryotic mRNA have polyA tails at their 3′-end. The RNA that does notcontain polyA is washed away and the bound RNA is recovered. The resultis an RNA sample enriched for the polyA-containing mRNAs. The polyA⁺ RNAis used as starting mRNA template for reverse transcription from anoligonucleotide primer. The oligonucleotide primer containspolydeoxythymidine (oligo dT) which can anneal to the poly A+ tail ofmRNA, and also preferably contains one or more restriction enzymecleavage sites for later use in cloning into vectors, and a further“END” sequence at the 5′-end of the primer that allows specificamplification.

[0021] The population of cDNA molecules generated by reversetranscription with a reverse transcriptase is heated to disrupt anysecondary structure, such as self-annealing, and then incubated understringent conditions to allow hybridization of complementary cDNAsegments. It is expected that a cDNA (antisense) complementarity to thesense cDNA will form an at least partially double-stranded hybridmolecule. cDNA generated from cis-encoded antisense mRNA, which arisesfrom transcription of the complementary strand of the sense gene, willhave a certain region of perfect complementarity to its correspondingsense cDNA. However, cDNA generated from trans-encoded antisense, whicharises from transcription of a different genomic locus, is expected tohave only partial complementarity to a sense cDNA from a differentgenomic locus. Even in such a case, functional trans-encoded antisensewill generate a double-stranded structure stable enough to be retainedby the enrichment method.

[0022] Treating the hybrid molecule, which is at least partiallydouble-stranded, with a DNA polymerase having 5′ to 3′ polymeraseactivity and 3′ to 5′ exonuclease activity produces double-strandedmolecules with complete complementarity. This treatment with DNApolymerase cleaves single-stranded DNA molecules with free 3′ ends untilthe DNA polymerase reaches a region of stable double-stranded structure,producing a DNA molecule with a double-stranded region and one or twoadjacent single-stranded DNA regions with free 5′ ends. This serves as atemplate for the 5′ to 3′ polymerase activity of the enzyme whichproduces a final product with blunt ends. The DNA polymerase having 3′to 5′ polymerase activity useful in the method according to the presentinvention is preferably T4 DNA polymerase. Four other enzymes useful inthe method according to the present invention are Platinum Pfx DNApolymerase and Deep Vent DNA polymerase (both from New England Biolabs,Beverly, Mass.), Pwo DNA polymerase (Roche) and Pfu DNA polymerase(Stratagene, La Jolla, Calif.). Other polymerases can be used, includingbut not limited to any suitable DNA polymerase having both aforesaidactivities.

[0023] The double-stranded molecules resulting from the DNA polymerasereaction are amplified by the polymerase chain reaction (PCR) using apolymerase, such as Taq DNA polymerase, or other thermostablepolymerases, and preferably with a primer identical to the END region ofthe poly dT-containing oligonucleotide primer previously used togenerate the population of cDNA. Thus, natural antisense mRNA moleculesas converted to double-stranded cDNA molecules are enriched. Theamplified double-stranded DNA molecule enriched for natural antisensemRNA can be easily cloned into a vector using one or more restrictionenzyme cleavage sites at the ends thereof (i.e., derived from theoligonucleotide primer). Confirmation that the cloned double-strandedcDNA encodes a natural antisense mRNA can be obtained by RT-PCR orNorthern blot analysis as described in the Example 1 herein.

[0024] The oligonucleotide primer used for amplifying thedouble-stranded molecules from the DNA polymerase reaction iscomplementary to and preferably identical to the END sequence located atthe 5′-end of the polydT primer for initially generating thedouble-stranded molecules. The location of the END sequence before (5′to) the restriction enzyme cleavage site(s) and polydT region) onlyamplifies the correct template and gives rise to products that have therestriction enzyme cleavage site(s) available for cloning. Thus, naturalantisense molecules can only be enriched and amplified if there is asuccessful polymerization which produces templates that can be amplifiedby the Endogenous Antisense Identification (EASI) procedure of thepresent invention.

[0025] Because natural antisense RNAs have been shown to play importantregulatory roles or provide new regulatory information for known genesin the control of cell growth, malignant transformation, and othercellular phenotypes, the present method provides a basis for finding newgenes with important cellular regulatory roles or new regulatoryinformation for known genes and provides a starting material fordevelopment of an antisense-based therapeutic to treat a disease ordisorder in which the down-regulation or inhibition of the sense gene ortranscript is sought.

[0026] The EASI method allows production of a cDNA population enrichedfor sequences representing endogenous antisense RNAs. In Example 1, oneapproach to use the method for the identification of the antisense RNAis described. This employs the derivation of cDNA libraries from theEASI enriched cDNA population, followed by sequencing of cDNA clones,characterization of the sequences, and analysis of the existence ofantisense RNA in the cells. Example 2 describes a way to use the EASIenriched cDNA population to derive probes for microarray analysis, whichallows both the detection of antisense transcripts and theirdifferential expression. While the specific RNA polymerase disclosed inExample 2 below is T7 RNA polymerase, this specific RNA polymerase canbe any bacteriophage RNA polymerase which uses DNA as template toproduce RNA, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNApolymerase, etc.

[0027] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration and is not intended to belimiting of the present invention.

EXAMPLE 1

[0028] The antisense enrichment procedure according to the presentinvention, also designated as the Endogenous Antisense Identification(EASI) procedure, was applied to a human glioma cell line and shown inexperiments to enrich for antisense mRNA/cDNA known to have a naturalantisense mRNA. The experimental results and the materials and methodsused in the experiments discussed in this example are provided below.

MATERIALS AND METHODS

[0029] cDNA First Strand Synthesis:

[0030] Two μg of poly A+ RNA from human A172 glioma cell line werediluted with double distilled water (DDW) up to 6 μl volume. Secondarystructure formation or self-annealing of RNA molecules is disrupted byincubation at 80° C. for 2 min and then quick chilled on ice. 4 μl of a10 mM oligo dT (deoxythymidine) primer, which contains cloning/cleavagesites for the NotI restriction enzyme and has the sequence5′-TTCTAGAATTCAGCGGCCGC(T)₁₈N_(1,(G,A,C))N_(2 (G,A,C,T))-3′ (SEQ IDNO:1) (where 5′-TTCTAGAATTCAGCGGCCGC-3′, corresponding to nucleotide 1to 20 of SEQ ID NO:1, is termed the “END” sequence), 4 μl of 5×Superscript buffer, 1 μl of RNasin, 2 μl of 0.1M DTT (dithiothreitol)and 2 μl of 10 mM dNTPs (deoxynucleoside 5′-triphosphates) were added tothe poly A+ RNA. After preheating at 42° C., 1 μl of RT Superscriptenzyme was added and incubated at 42° C. for 1.5 hours. The reactionmixture was then heated to 80° C. for 2 min and the RNA strands weredigested with alkaline treatment, followed by neutralization.

[0031] Hybridization:

[0032] The cDNA generated was precipitated and then resuspended with 30μl hybridization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, pH 8.0, 0.4 MNaCl, 80% formamide, DDW). Any secondary structure was disrupted at 85°C. for 10 min and then the cDNA was rapidly transferred to a 52° C. bathfor 16 hours. After 16 hours of incubation, the cDNA was precipitatedwith ethanol and resuspended in 30 μl DDW.

[0033] T4 DNA Polymerase Treatment:

[0034] Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 μl of hybridized cDNA molecules with T4 DNA Polymerase (3-6 units),dNTPs, T4 reaction buffer, and BSA (bovine serum albumin) to a finalvolume of 80 μl. After reacting for 1 hour at 16° C., 5 μl of Klenowsequencing grade enzyme was added and then transferred for 30 min at anincubation temperature of 37° C. The inactivation of the enzymes wascarried out at 75° C. for 10 min, followed by phenol/chloroformextraction and EtOH precipitation. The precipitated pellet wasresuspended in 12 μl of DDW for use as the PCR template in the next PCRamplification step.

[0035] Platinum Pfx DNA Polymerase Treatment

[0036] Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 1 ml of Platinum Pfx DNApolymerase (2.5 u/ml), 1.5 ml 10 mM dNTPs, 5ml 10× Pfx Amplificationbuffer, 1 ml 50 mM MgSO2 and 11.5 ml DDW to a final volume of 50 ml andthen transferred for incubation at 72° C. for 5 min. The inactivation ofthe enzymes was carried out at 75° C. for 10 min, followed byphenol/chloroform extraction and EtOH precipitation. The precipitatedpellet was resuspended in 12 ml of DDW for use as the PCR template inthe next PCR amplification step.

[0037] Deep Vent DNA Polymerase Treatment

[0038] Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 2 ml 10 mM dNTPs, 5 ml 10×Thermo polymerase reaction buffer, 2 ml 100 mM MgSO2 and 10 ml DDW to avolume of 49 ml and preincubated at 72° C. for 2 min. 1 ml of Deep VentDNA polymerase (2 u/ml) was added and the whole mixture was transferredfor incubation of the reaction at 72° C. for 5 min. The inactivation ofthe enzymes was carried out at 75° C. for 10 min, followed byphenol/chloroform extraction and EtOH precipitation. The precipitatedpellet was resuspended in 12 ml of DDW for use as the PCR template inthe next PCR amplification step.

[0039] Pwo DNA Polymerase Treatment

[0040] Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 2 ml 10 mM dNTPs, 5 ml 10×PCRbuffer with 20 mM MgSO2 and 12 ml DDW to a volume of 49 ml and thentransferred for preincubation at 72° C. for 2 min. Then 1 of Pwo DNApolymerase (5 u/ml) was added and the whole mixture was transferred forincubation at 72° C. for 5 min. The inactivation of the enzymes wascarried out at 75° C. for 10 min, followed by phenol/chloroformextraction and EtOH precipitation. The precipitated pellet wasresuspended in 12 ml of DDW for use as the PCR template in the next PCRamplification step.

[0041] Pfu DNA Polymerase Treatment

[0042] Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 5 ml cloned Pfu DNA polymerase(2.5 u/ml), 5 ml 10 mM dNTPs, 5 ml 10× cloned Pfu polymerase buffer, and5 ml DDW to a final volume of 50 ml and then transferred for incubationat 72° C. for 5 min. The inactivation of the enzymes was carried out at75° C. for 10 min, followed by phenol/chloroform extraction and EtOHprecipitation. The precipitated pellet was resuspended in 12 ml of DDWfor use as the PCR template in the next PCR amplification step.

[0043] PCR Amplification:

[0044] 1 μl from the resuspended DNA pellet resulting from the T4 DNApolymerase treatment was used as a template for PCR amplification withthe same END primer derived from the oligo dT primer used for theinitial cDNA synthesis. The PCR conditions are as follows: The reactionwas performed in a total volume of 50 μl with 1 μl template afterblunting (T4 DNA polymerase treatment), 5 μl Boehringer 10× buffer 2, 1μl 10 mM dNTPs, 4 μl 10 μM primer, 0.5 μl PERFECT MATCH PCR enhancer(Stratagene) La Jolla, Calif., 0.5 μl of (3.5 u/μl) Boehringer Taq highfidelity DNA polymerase (Boehringer-Mannheim) and DDW for the remainingvolume. The temperature program used for PCR was as follows: 2 min of94° C. for denaturation and 16 cycles of: denaturation at 94° C. for 30sec, annealing at 64° C. for 30 sec, and extension at 72° C. for 4 min.At the end of the cycling, the extension was completed at 72° C. for 7min.

[0045] Cloning:

[0046] The PCR amplified product was digested with NotI and ligated intopBLUESCRIPT vector (Stratagene) linearized with the restriction enzymeEag1. EagI-cleaved sticky ends are compatible with NotI-cleaved stickyends.

[0047] The transformants were detected in the presence of X-Gal(5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) and IPTG (isopropylβ-D-thiogalactopyranoside). White colonies containing inserts weresequenced following miniprep DNA purification. All the sequencingproducts were analyzed by common Bioinformatic programs, such as BLAST,and the interesting sequences were used as templates for single-strandedprobes for Northern blot analysis to confirm the presence of anantisense molecule.

[0048] A real antisense is determined to be present by use of tworeverse complementary single-strand (ss) probes, each complementary to adifferent strand. For a known gene, the ss probe complementary to thesense strand will hybridize to the sense mRNA and give a signal on aNorthern blot to a band of expected size. The ss probe complementary tothe antisense strand will give a signal on a Northern blot if anantisense mRNA is present in the RNA population.

[0049] Reverse Transcription—PCR Amplification

[0050] The following gene-specific RT-PCR conditions were found to be ofhigh specificity to the targeted gene. Poly A+ selected mRNA (100 ng)was used as template and the reactions were carried out with theThermoscript RT enzyme (100 ng polyA+) (Gibco/BRL). RNA, 4 μl of 5×buffer, 10 μM primer and H₂O were mixed to a final volume of 14 μl. Themixture was incubated at 85° C. for 1 minute and then at 65° C. for 5minutes. The following were then added while the tube was held at 60°C.: 1 μl of 0.1 mM DTT, 1 μl of RNAseOut (40 units/μl), 2 μl of 10 mMdNTPs and 1 μl H₂O. 1 μl of Thermoscript RT (15 units/μl) was added andthe reaction was carried out at 60° C. For 1 hour. The reaction was thenterminated by incubation at 85° C. For 5 minutes with the RNA beingremoved by the addition of 1 μl of RNAseH (1 u/μl; Boehringer) andincubation at 37° C. For 20 minutes.

[0051] The PCR amplication reaction was conducted under standardconditions using a pair of sense and antisense primers for the testedgene. Taq DNA polymerase from Boehringer was used. The initial cycleconsisted of incubation at 94° C. For 3.5 minutes followed by 5 cyclesof: denaturation at 94° C. For 30 seconds, annealing at 62° C. For 30seconds and extension at 72° C. For 2 minutes. This was then followed by25 cycles of: 94° C. For 30 seconds, 58° C. For 30 seconds and 72° C.For 2 minutes. The reaction was terminated with a step of incubation at72° C. For 7 minutes.

[0052] Primers and Sequences

[0053] The primers and sequences used in the EASI procedure describedand exemplified in this example are provided below. Human c-erbERB-SEN2: 5′-GATGGGAGTTGTGTGTTTAGTC-3′ (SEQ ID NO:2) ERB-RC:5′-GGAGAGAGAAGTGCAGAGTTCG-3′ (SEQ ID NO:3) Human ku autoantigen KU_FOR:5′-TTAGTACAAACTTAGGGCTCT-3′ (SEQ ID NO:4) KU_REV:5′-TCATGGCAACTCCAGAGCAG-3′ (SEQ ID NO:5) Human GAPDH GAPDH_FOR:5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO:6) GAPDH_REV:5′-TCCACCACCCTGTTGCTGTA-3′ (SEQ ID NO:7) Human RbAp48 (clone N1-15)N1-15FOR: 5′-GGAGTTAGTCCTTGACCACTAG-3′ (SEQ ID NO:8) N1-15RC:5′-GCACTTACACAGTTAGTCATGG-3′ (SEQ ID NO:9) Sequence of clone N1-15-designated SEQ ID NO:10

[0054] Northern Hybridization Using Single-Stranded Probe

[0055] For each interesting fragment that was obtained from thebioinformatic analysis, two primers, forward and reverse, that aresuitable for the two ends of the fragment were prepared. Northern blotswere prepared according to CURRENT PROTOCOLS IN MOLECULAR BIOLOGY by F.M. Ausubel et al, Chapter 4.a with 6 μg of poly A+ RNA per lane.

[0056] Synthesis of Labeled Single-Stranded DNA Probes:

[0057] The cDNA insert of the tested gene (isolated by digestion or byPCR from the plasmid) was labeled using well-known labeling reactionswith Klenow enzyme. A specific sense or antisense primer was used todrive the synthesis of the probe in the presence of radioactivenucleotides. Thus, only one strand (sense or antisense, depending on theprimer used) was synthesized and labeled with radioactivity. For eachgene, the sense and antisense probes were generated and used forhybridization on separate blots.

[0058] For the preparation of DNA template, 0.5-1 μg of template from aminiprep DNA isolation procedure was denatured with NaOH treatment andincubated for 5 min. at RT (room temperature), precipitated with NH₄AcpH 5.2, yeast tRNA and 100% EtOH. After ethanol precipitation, thepellet was redissolved in 7 μl of DDW.

[0059] For the synthesis of labeled single strands, 50-100 ng of thedenatured template DNA was mixed and annealed with 10 pmols of theappropriate primer in Klenow buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂,7.5 mM DTT), and incubated at two different temperatures, 15 min at 60°C. and then 15 min. at RT. α-³²P-dCTP (specific activity of 3000ci/mmol), Klenow enzyme (5 units) and dATP, dTTP and dGTP nucleotidesolution (0.5 mM final concentration) were then added to the annealingmix and incubated for 20 min. at 37° C. to incorporated label into thesynthesized strand of DNA. Afterwards, “cold” (non-radioactive) dCTP wasadded and the reaction was further incubated for 15 min. at 37° C. Theinactivation of the Klenow enzyme was carried out by incubation at 75°C. For 10 min. The purification of the probe was done on a SEPHADEX G-50column.

[0060] Northern Blot Hybridization and Post-Hybridization Processing

[0061] 1-5×10⁶ cpm of denatured labeled probe was added per 1 mlhybridization solution and incubated overnight at 65° C.

[0062] Prehybridization was carried out for 30 min to 2 hours at 65° C.The Northern blot was washed 3 times in 2×SSC, 0.2% SDS for 7 min. at RTand then twice with high stringency washing solution that contain0.1×SSC, 0.2% SDS for 15 min. at 60° C. The blot was then exposed ontoX-Ray films.

RESULTS AND DISCUSSION

[0063] The antisense enrichment procedure was applied to examine thepossible involvement of antisense RNAs in the response of the humanglioma cell line A172 to hypoxia stress. This is a cell line model forthe response of glial cells in the brain to ischemic injury that canresult from events such as stoke. To detect antisense mRNAs that areinvolved in the response of the cells to hypoxia conditions, poly A+selected mRNA was obtained from cells grown under hypoxia conditions aswell as from cells grown under normal conditions. Single-stranded cDNAprepared from the two mRNA pools were mixed in equal proportions andused as the basis for the antisense enrichment procedure.

[0064] A gene, known to have an endogenous antisense mRNA was comparedto two genes, for which no endogenous antisense mRNA is known to exist,to test the ability of the antisense enrichment procedure to enrich forantisense cDNAs. The presence of these three genes was tested before andafter application of a preferred embodiment of the antisense enrichmentmethod according to the present invention. As shown in FIG. 2, PCRamplification was performed for each gene with two gene specificprimers, (1) before the antisense enrichment procedure on single strandcDNA (RT), and (2) is known to on DNA obtained after antisenseenrichment (AS). The c-erb cDNA is known to be found at very low levelsin the mRNA and gave no signal after PCR of normal RT products (20 ngpoly A+ selected RNA). However, using a similar amount of DNA template,a strong signal was obtained from DNA obtained from the antisenseenrichment procedure. The two other genes, Ku and GAPDH, for which thereare no known antisense mRNA, and which are expressed at much higherlevels, show a clear decrease in abundance in FIG. 2. This indicatesthat the antisense enrichment procedure according to the presentinvention advantageously enriches for cDNA for which a natural antisensecounterpart exists (c-erb) in contrast to cDNA which lacks a naturalantisense counterpart (Ku, GAPDH).

[0065] The final products of the antisense enrichment procedure werecloned as described in the materials and methods section and a librarywas created. After sequencing and bioinformatics analysis, few of theclones which matched known genes were chosen for further analysis todetermine if they originated from an endogenous antisense mRNA. In orderto examine the presence of antisense mRNAs to a specific gene, a genespecific RT-PCR method was established in which gene specific primerswere used to initiate the reverse transcription reaction. To detect thenormal (sense) transcript of a gene, an antisense primer was used.Conversely, when a sense primer was used, it should be possible todetect antisense mRNAs. It was found that standard RT-PCR reactions werenot specific enough, and when a gene specific primer, either sense orantisense, was used, amplification products, for which no antisensetranscript was known to exist for the gene, was observed. Moreover,other genes were also amplified. This indicates that standard RT-PCRreaction conditions resulted in non-specific reverse transcription ofother mRNAs. A gene specific RT-PCR procedure was then established usinghighly specific conditions described in the Materials and Methodssection of this example to confirm if the clones originated from naturalantisense RNA. A highly specific RT reaction was performed with a senseprimer (relative to the known sense mRNA) using Thermoscript reversetranscriptase (Gibco BRL).

[0066] Under the aforesaid highly specific conditions such a reactionwill result in a PCR product only if an antisense mRNA exists. This wascompared to an RT reaction done using the antisense primer. Table 1includes the list of clones from the antisense enriched library thatwere individually confirmed for the presence of matching antisense RNA.One clone, clone N1-15 (RbAp48 mRNA encoding retinoblastoma bindingprotein, accession number X74262), obtained from the antisenseenrichment-derived library, is shown here in detail. A primer pair(sense and antisense primers) specific for the sequence obtained in thelibrary was synthesized. Each primer was used to derive a RT reactionusing Thermoscript reverse transcriptase. As a control, the same wasdone for GAPDH for which there is no known antisense mRNA. For bothgenes, the sense primer (S) is expected to support reverse transcriptionof antisense mRNA while the antisense primer (AS) is expected to supportthe synthesis of the sense (normal) mRNA. In FIG. 3, a clear signal wasobtained with the sense primer (S) derived RT-PCR of the N1-15 clonewhereas none was obtained (as expected) for the GAPDH mRNA. Theantisense (AS)-derived RT-PCR however gave the expected products. Thisdemonstrates that for clone N1-15, which matches the RbAp48 mRNAencoding retinoblastoma binding protein, PCR products of the expectedsize were obtained for both sense and antisense RT-PCR, whereas thecontrol gene, GAPDH, which does not have any known antisense mRNA,resulted in a product only with the antisense primer. This suggestedthat an endogenous antisense mRNA does exist for this N1-15 gene. TABLE1 Strand- Clone Insert specific Northern Blots Bio name Gene nameAccession length Match location % identity RT-PCR Sense Antiseninformatics N1_15 RbAp48 encoding NM_005610 169  2179-2307 100 Positive3.0 Kb 3.5 Kb Positive retinoblastoma binding protein N1_27 CD9 antigenNM_001769 550  621-1171 99 Positive ND ND N1_33 Thymosin b-10 S54005 491  1-447 97 Positive 0.6 Kb none N2_20 Uncharacterized AF187554 170 555-669 100 Negative* 7.0 Kb 0.5 Kb N2_66 2-19 gene (downstream toX55448 206 42663-42866 99 Positive G6PD) N3_13 Calumein AF013759 143 2273-2415 100 Positive ND ND D1-1 SMRTE, Silencing AF125672 111 8573-8667 96 Positive 8.5 kb 3 kb mediator of retinoic acid and thyroidhormone receptor a D1-19 M-phase phosphoprotein, X98260 100  578-661 98Negative ND ND NO Antisense mpp11 D1-48 Metallothionein 2 X97260 189 225-327 98 Positive ND ND D2-31 Integrin a 3 NM_005501 94  4421-4495 98Positive 4.5 kb 4.5 kb D2-3 S100 calcium-binding NM_002966 161  549-649100 Negative ND ND NO Antisense protein A10 D2-7 Regulator of G-proteinAF030108 106  340-436 100 Negative ND ND NO Antisense signaling 5 (RGS5)D2-20 14-3-3n L20422 50  1524-1557 100 Negative ND ND NO AntisenseN26_3_50 Ribosomal protein S11 X06617 586  538-1 99 Positive ND NDPositive Antisense Human isocitrate U52144 gene dehydrogenase mRNAN2_3_40 Homo sapiens ribosomal NM_001030 363   1-329 (29-357) 99Positive ND ND Positive protein S27 (metallopanstimulin 1) (RPS27)Antisense S24A Human ribosomal M31520  249-353 (116-12) 100 gene proteinS24 mRNA N3_45 UbA52 placental mRNA X56999 563   52-552 99 Positive NDND for Ubiquitin-52 fusion protein 25_3_49 Homo sapiens cDNA AK000501662  573-351: 352-24 95 ND ND ND Positive FLJ20494 fis, clone KAT08547M.musculus mRNA for Y08702 504  449-329: 330-236: 86 neuronal protein15.6  186-104 20_2_33 Homo sapiens actin, beta NM_001101  1326-1622(19-315) 97 Positive ND ND (ACTB) mRNA Matches  1688-1791 to normal mRNAand (382-485) to pseudogene.

[0067] In order to determine if such an endogenous antisense mRNA indeedexists for the RbAp48 gene, a Northern blot analysis using sense andantisense single-stranded probes was performed. RNA from human A172glioma cells grown under normal conditions or under hypoxia for 4 or 16hours were separated by eleotrophosis on formaldehyde-agarose gels andblotted onto nylon membranes. One blot was hybridized with an antisensesingle stranded probe which should reveal the existence of any antisenseRNA and a second blot was hybridized with the antisense probe to revealthe normal sense mRNA. As shown in FIG. 4, specific bands of differentsize hybridized with the different probes. These results demonstrate theexistence of a mRNA species that hybridizes specifically with the senseprobe and which is of a different size from the normal sense mRNA andfurther indicate that an endogenous antisense mRNA indeed exists for theRbAp48 gene. Interestingly, an inverted correlation is observed in theexpression of the sense and antisense RbAp48 mRNAs under hypoxia, wherethe sense mRNA is reduced while the antisense mRNA is induced to higherlevels, suggesting a regulatory role for the antisense RNA.

[0068] As can be seen from Table 1, 14 of the 18 tested clones werefound to be positive under the experimental conditions or bybioinformatic analysis (clone 25-3-49). The bioinformatic analysisutilized the BLAST program (NCBI) to find matches between the clone anda certain region of another gene that is complementary to it. Theanalysis thus showed that the “EASI” antisense enrichment methodology isefficient.

[0069] Table 2 identifies the inserts in the clones listed in Table 1 bySEQ ID NO. TABLE 2 Insert from clone SEQ ID NO: N1_15 11 N1_27 12 N1_3313 N2_20 14 N2_66 15 N3_13 16 D1-1 17 D1-19 18 D1-48 19 D2-31 20 D2-3 21D2-7 22 D2-20 23 N26-3-50 24 N2-3-40 25 N3-45 26 25-3-49 27 20-2-33 28

EXAMPLE 2

[0070] To produce probes for microarray analysis from the EASI enrichedcDNA, the following procedure may be used (graphically represented inFIG. 5) RNA is derived from cell population A (A-RNA) and from cellpopulation B (B-RNA). A reverse transcription reaction with regularoligo-dT is used to make single stranded cDNA from A-RNA (A-sscDNA). ForB-RNA an oligo-dT that has an additional sequence of the T7 RNApolymerase promoter region is used (e.g., SEQ ID NO:29). This produces asingle stranded cDNA population (B-sscDNA) that has the T7 RNApolymerase promoter (T7RPP) region at its 5′ end. Using the regular EASImethod described in Example 1, the A-sscDNA and B-sscDNA populations aremixed, and allowed to anneal to each other. A DNA polymerase that alsohas a 3′ to 5′ exonuclease activity is then used (e.g., T4 DNApolymerase) to enrich for the population of cDNA that annealed to eachother. The result of this reaction is a chimeric double stranded cDNAthat has the T7 RPP on one of its ends. This now becomes the templatefor a T7 RNA polymerase reaction. The RNA produced from this reactioncan now be labeled with a fluorescent dye for DNA microarray analysisusing standard methods.

[0071] It should be noted that only in the cases where an antisense RNAis over-expressed in one population (e.g., A-RNA), and its complementarysense mRNA is over-expressed in the second population (e.g., B-RNA),will the T7RPP be on only one end of the double stranded cDNA template.If sense and antisense RNAs are found in the same population (B-RNA),they will anneal to each other and the resulting double stranded cDNAwill have the T7 RNA polymerase promoter region on both ends. Such atemplate will cause production of both sense and antisense RNA that willcancel each other during hybridization. Thus, significant labeling isproduced only in cases where there is differential expression of theantisense RNAs.

[0072] For hybridization to DNA microarrays, the EASI T7RP derived RNAcan be labeled with one fluorescent die (e.g., Cy3). This probe can becompared to a probe derived from A-RNA or from B-RNA. Such a probe willbe labeled with another fluorescent dye (e.g., Cy5). After hybridizationto the DNA microarray, differentially expressed antisense RNA can bedetected by simple analysis of the hybridization results. Since the EASIT7RP derived probe enriches for cases in which there is differentialexpression of antisense RNA, the enrichment will be observed by a highersignal from the Cy3 label when compared to the Cy5 label.

[0073] One limitation of this approach is that the analysis cannotdisclose the pattern of differential expression, namely, in whichpopulation is sense over-expressed and in which is antisenseover-expressed. The following provides one way to circumvent thislimitation. After the production of sscDNA from B-RNA, the 3′ end isblocked by the addition of a nucleotide analog such as deoxynucleoside[1-thio] triphosphate. Such an analog can be added by using the enzymeterminal deoxy transferase (TdT) or by ligating a primer that includesdeoxynucleoside [1-thio] triphosphate analogs, to the 3′ end with anappropriate ligase (e.g., T4 RNA ligase) that also ligatesoligonucleotides to single stranded DNA. Another option is toincorporate such analogs into the sscDNA during the reversetranscription reaction. Once such modified sscDNA, carrying the T7RPP onits 5′ end, is produced, it will be resistant to the exonucleaseactivity of the T4 DNA polymerase during the subsequent EASI reaction.Using this modification approach only sscDNA templates which areunmodified, coming from the A-RNA population, will be templates for theEASI procedure. Hybrids of sense and antisense cDNA derived from B-RNAwill be completely resistant to the EASI procedure and will not berepresented in the enriched population of EASI cDNA (FIG. 7). The onlypopulation of cDNA that will be meaningful is the one that has the T7RPPon one side (FIG. 6).

[0074] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

[0075] While this invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations of the inventions following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth as follows in the scope ofthe appended claims.

[0076] All references cited herein, including journal articles orabstracts, published or corresponding U.S. or foreign patentapplications, issued U.S. or foreign patents, or any other references,are entirely incorporated by reference herein, including all data,tables, figures, and text presented in the cited references.Additionally, the entire contents of the references cited within thereferences cited herein are also entirely incorporated by references.

[0077] Reference to known method steps, conventional methods steps,known methods or conventional methods is not in any way an admissionthat any aspect, description or embodiment of the present invention isdisclosed, taught or suggested in the relevant art.

[0078] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying knowledge within the skill of the art (including the contentsof the references cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. In is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

[0079] Bedford M, Arman E, Orr U A and Lonai P (1995) Analysis of theHoxd-3 gene: structure and localization of its sense and naturalantisense transcripts. DNA Cell. Biol. 14: 295-304.

[0080] Chang Y, Spicer D B and Sonenshein G E (1991) Effects of IL-3 onpromoter usage, attenuation and antisense transcription of the c-myconcogene in the IL-3-dependent Ba/F3 early pre-B cell line. Oncogene 6:1979-1982.

[0081] Farrell C M and Lukens L N (1995) Naturally occurring antisensetranscripts are present in chick embryo chondrocytes simultaneously withthe down regulation of the alpha 1 (I) collagen gene. J. Biol. Chem.270: 3400-3408.

[0082] Fu X D and Maniatis T (1992) Isolation of a complementary DNAthat encodes the mammalian splicing factor SC35. Science 256: 535-538.

[0083] Khochbin S and Lawrence J (1989) An antisense RNA involved in p53mRNA maturation in murine erythroleukemia cells induced todifferentiate. EMBO J. 8: 4107-4114.

[0084] Kimelman D and Kirchner M W (1989) An antisense mRNA directs thecovalent modification of the transcript encoding fibroblast growthfactor in Xenopus oocytes. Cell 59: 687-696.

[0085] Krystal G W, Armstrong B C and Battey J F (1990) N-muc mRNA formsan RNA-RNA duplex with endogenous antisense transcripts. Mol. Cell. Bio.10: 4180-4191.

[0086] Lankenau S, Corces V G and Lankenau D H (1994) The Drosophilamicropia retrotransposon encodes a testes specific antisense RNAcomplementary to reverse transcriptase. Mol. Cell. Biol. 14: 1764-1775.

[0087] Lee R C, Feinbaum R L and Ambros V (1993) The C. elegansheterochronic gene lin-4 encodes small RNAs with antisensecomplementarity to lin-14. Cell 75: 843-854.

[0088] Michael N L, Vahey M T, d'Arcy L Ehrenberg P K, Mosca J D,Rappaport J and Redfield R R (1994) Negative-strand RNA transcripts areproduced in human immunodeficiency virus type 1 infected cells andpatients by a novel promoter down-regulated by Tat. J. Virol. 68:979-987.

[0089] Murphy P R and Knee R S (1994) Identification andcharacterization of an antisense RNA transcript (gfg) from the humanbasic fibroblast growth factor gene. Mol. Endocrinol. 8: 852-859.

[0090] Noguchi M, Miyamoto S, Silverman T A and Safer B (1994)Characterisation of an antisense Inr element in the eIF-2a gene. J.Biol. Chem. 269: 29161-29167.

[0091] Vanhee-Brossollet C and Vaquero C (1998) Do natural antisensetranscripts make sense in eukaryotes? Gene 211: 1-9.

1 29 1 40 DNA Artificial Sequence Synthetic oligonucleotide primer 1ttctagaatt cagcggccgc tttttttttt ttttttttvn 40 2 22 DNA ArtificialSequence Synthetic oligonucleotide primer 2 gatgggagtt gtgtgtttag tc 223 22 DNA Artificial Sequence Synthetic oligonucleotide primer 3ggagagagaa gtgcagagtt cg 22 4 21 DNA Artificial Sequence Syntheticoligonucleotide primer 4 ttagtacaaa cttagggctc t 21 5 20 DNA ArtificialSequence Synthetic oligonucleotide primer 5 tcatggcaac tccagagcag 20 620 DNA Artificial Sequence Synthetic oligonucleotide primer 6 accacagtccatgccatcac 20 7 20 DNA Artificial Sequence Synthetic oligonucleotideprimer 7 tccaccaccc tgttgctgta 20 8 22 DNA Artificial Sequence Syntheticoligonucleotide primer 8 ggagttagtc cttgaccact ag 22 9 22 DNA ArtificialSequence Synthetic oligonucleotide primer 9 gcacttacac agttagtcat gg 2210 188 DNA Artificial Sequence PCR Amplified Sequence 10 gggcgggccgcttttttttt tttttttttg gagttagtcc ttgaccacta gtttgatgcc 60 atctccattttgggtgacct gtttcaccag caggcctgtt actctccatg actaactgtg 120 taagtgcttaaaatggaata aattgctttt ctacataacc ccaaaaaaaa aaaaaaaaaa 180 gcggccgc 18811 169 DNA Artificial Sequence PCR Amplified Human 11 ttttttttttttttttttgg agttagtcct tgaccactag tttgatgcca tctccatttt 60 gggtgacctgtttcaccagc aggcctgtta ctctccatga ctaactgtgt aagtgcttaa 120 aatggaataaattgcttttc tacataaccc caaaaaaaaa aaaaaaaaa 169 12 550 DNA ArtificialSequence PCR Amplified Human 12 ttttcattgt cataattttt tattatgtatcaaattgtct tcaatataag ttacaacttg 60 attaaagttg atagacattt gtatctatttaaagacaaaa aaattctttt atgtncaata 120 tcttgtctag agtctagcaa atatagtacctttcattgca ggatttctgc ttaatataac 180 aagcaaaanc aaacaactga aaaaatataaaccaaagcaa accaaacccc ccgctcaact 240 acaaatgtca atattgaatg aagcattaaaagacaaacat aaagtaactt cagcttttat 300 ctagcaatgc agaatgaatn ctaaaattagnggcaaaaaa ncaaacaaca aacaacaaac 360 aaaacaaanc aaacaancaa aaaatcccaccaatcttcat gggtaaactt tcctgctcag 420 ggatgtaagc tgactctaga ccattngcggttcctgcgga tagcacagcc angatcatct 480 gaagatcatg ccaaatntca tgaccacggcaatgccgatg cccctgcgcc gatgatgngg 540 aatttattgg 550 13 491 DNAArtificial Sequence PCR Amplified Human 13 tttttttttt tttttttcttgctgcagcaa cgcgagtggg agcaccagga tctcgggctc 60 ggaacgagac tgcacggattgttttaagaa aatggcagac aaaccagaca tgggggaaat 120 cgccagcttc gataaggccaagctgaagaa aacggagacg caggagaaga acaccctgcc 180 gaccaaagag accattgagcaggagaagcg gagtgaaatt tcctaagatc ctggaggatt 240 tcctaccccc atcctcttcgagaccccagt cgtgatgtgg aggaagagcc acctgcaaga 300 tggacacgag ccacaagctgcactgtgaac ctgggcactc cgtgccgatg ccaccggcct 360 gtgggtctct gaagggacccccccccaatc ggactgccaa attctccggt ttgccccggg 420 atattataga aaattatttgtatgaataat gaaaataaaa cacacctcgt ggcaaaaaaa 480 aaaaaaaaaa a 491 14 206DNA Artificial Sequence PCR Amplified Human 14 tttttttttt ttttttttgggagtggtagg atgaaacaat ttggagaaga tagaagtttg 60 aagtggaaaa ctggaagacagaagtacggg aaggcgaaga aaagaataga gaagataggg 120 aaattagaag ataaaaacatacttttagaa gaaaaaagat aaatttaaac ctgaaaagta 180 ggaagcagaa aaaaaaaaaaaaaaaa 206 15 206 DNA Artificial Sequence PCR Amplified Human 15ttttctgtgg ggccatcact ttattaaggg gtcatctaga aggtgggccc cctgncaaac 60cgcgggactg tgatcgggct ccagctactt caccaccccg ggccagcctg ctccaggggt 120cccttcctgc tgagagcagg cgagaggcag tcaggctcat gaagcagcca ccgggtttgg 180ctcactggaa ggaatcacac tggaaa 206 16 178 DNA Artificial Sequence PCRAmplified Human 16 tttttttttt ttttttttct gtgtccactg gagagcttgagctcacactc aaagatcaga 60 ggacctacag agagggctct ttggtttgag gaccatggcttacctttcct gcctttgacc 120 catcacaccc catttcctcc tctttccctc tccccgctgccaaaaaaaaa aaaaaaaa 178 17 127 DNA Artificial Sequence PCR AmplifiedHuman 17 gaattcgatg cgtattctgt ggcccgccat ctgcgcaggg tggtggtattctgccattta 60 cacacgtcgt tctaattaaa aagcgaatna tactccaaaa aaaaaaaaaaangcggccgt 120 tgaattc 127 18 115 DNA Artificial Sequence PCR AmplifiedHuman 18 gaattcagcg gccgcttttt tttttttttt tcttcgaagt gtttaccccagtgtttgaaa 60 gggattccag atggtcaaat aaaaaaaatg ttcctaaact tggtgatatgaactc 115 19 204 DNA Artificial Sequence PCR Amplified Human 19gaattcaggg ccgttctggt tctctctntc tccccgccct ccctcaccac cagtggaacc 60ttcatcgagt tccacaaacc tggatttttt atgtacaacc ctgaccgtgg ccgtttgcta 120tattcctttt tctatgaaat aatgtgaatg ataataaaac agctttgact tgaaaaaaaa 180aaaaaaaaag cggccgctga attc 204 20 109 DNA Artificial Sequence PCRAmplified Human 20 gaattccctc cccctccttg tgccttcttt gtatataggcttctcacggc gaccaataaa 60 cagctcccag tttgtatgca aaaaaaaaaa aaaagcggccgctgaattc 109 21 191 DNA Artificial Sequence PCR Amplified Human 21gaattcagcg gccgcttttt tttttttttt ttgggagaag tgtataaatt attatgttga 60caagcagaga aagaaaagtt aaataccaga taagcttttg atttttgtat tgtttgcatc 120cccttgccct caataaataa agttcttttt tagttccaaa aaaaaaaaaa aaaaaagcgg 180ccgctgaatt c 191 22 106 DNA Artificial Sequence PCR Amplified Human 22gaattcagcg gaaaaccttg agttctggat tgcctgtgag gattacaaga agatcaagtc 60ccctgccaag atggctgaga aggcaaagca aatttatgaa gaattc 106 23 63 DNAArtificial Sequence PCR Amplified Human 23 gaattcaatg ggtaaataaatgctgctttg gggaaaaaaa aaaaaaaagc ggccgctgaa 60 ttc 63 24 586 DNAArtificial Sequence PCR Amplified Human 24 tttttttttt tttttttggcctgggaatga gaaaataact ttatttcatt gtggggagcg 60 ggccgatgtc cagcctcagaacttctggaa ctgcttcttg gtgccggcag ccttggtgac 120 cttgagcacg ttgaagcgcactgtcttgct cagaggccgg cactcgccca ctgtgacgat 180 gtcaccgatc tggacgtccctgaagcaggg ggacaggtgt acagacatgt tcttgtggcg 240 cttctcgaag cggttgtacttgcggatgta gtgcagatag tctcggcgga tgacaatggt 300 cctctgcatc ttcatcttggtcaccacgcc agagaggatc cgccctcgaa tggacacatt 360 accaagtgaa ggggcatttcttgtcaatgt aggtgccctc aatagcctcc ttgggtgtct 420 tgaagcccag accgatgttcttgtagtacc gcgggagctt ctccttgcca gtttctccca 480 gcaggaccct cttcttgttttgaaagatgg tcggctgctt ttggtangca cgctcagtct 540 gaatgtccgc catcttcccgggcgcctgaa aaaaaaaaaa aaaaaa 586 25 363 DNA Artificial Sequence PCRAmplified Human 25 tttttttttt ttttttttcc ggcggtgacg acctacgcacacgagaacat gcctctcgca 60 aaggatctcc ttcatccctc tccagaagag gagaagaggaaacacaagaa gaaacgcctg 120 gtgcagagcc ccaattccta cttcatggat gtgaaatgcccaggatgcta taaaatcacc 180 acggtcttta gccatgcaca aacggtagtt ttgtgtgttggctgctccac tgtcctctgc 240 cagcctacag gaggaaaagc aaggcttaca gaaggatgttccttcaggag gaagcagcac 300 taaaagcact ctgagtcaag atgagtggga aaccatctcaataaacacat tttggataaa 360 ccg 363 26 563 DNA Artificial Sequence PCRAmplified Human 26 tttttttttt tttttttctt cagcgaggcg gccgagctggttggtggcgg cggtcgtgcg 60 gacgcaaaca tgcagatctt tgtgaagacc ctcactggcaaaaccatcac ccttgaggtc 120 gagcccagtg acaccattga gaatgtcaaa gccaaaattcaagacaagga gggtatccca 180 cctgaccagc agcgtctgat atttgccggc aaacagctggaggatggccg cactctctca 240 gactacaaca tccagaaaga gtccaccctg cacctggtgttgcgcctgcg aggtggcatt 300 attgagcctt ctctccgcca gcttgcccag aaatacaactgcgacaagat gatctgccgc 360 aagtgctatg ctcgccttca ccctcgtgct gtcaactgccgcaagaagaa gtgtggtcac 420 accaacaacc tgcgtcccaa gaagaaggtc aaataaggtggttctttcct tgaagggcag 480 cctcctgccc aggccccgtg gccctggagc ctcaataaagtgtccctttc attgactgga 540 gcagcaaaaa aaaaaaaaaa aaa 563 27 662 DNAArtificial Sequence PCR Amplified Human 27 tttttttttt ttttttttgggactttcagc ccctttaatt aggtgctctg agaagaggtc 60 agaatggcag gcagggggtggggaaggcgg tgcttcttga gccccactta gcaactggtc 120 actcatcctc tggcagctggatcttgctgg ggtcgaagca gttggattcc atgatgggaa 180 ggccattggc ctctcggtatttcacaagcc tctcagcttc gcggcgggac cactctttca 240 tcccatccca cgctcttggacaccctgtgc acctgtagtc aggcagatag gccacaaagg 300 tgctgccaag gaccangatgatggagacgc caaagaagaa gacaagtcgc atgttccaaa 360 cgtccaaaaa cgggggccctgtcataacca atggggaatc cggggtcctc ccatacaagt 420 tttcgtcctc gggttctgggtcctcttgcc acggtgtggt cggttctggg ggccgctttc 480 ccgccacagc ggacggggcgaccacaatcc tggagaaact agattcccaa cgggacgccg 540 gcgggccggg aaccctcgcgtcgccgctgc cgccaaaaga ccgngaacgc tcaaccaaac 600 agccaaccgc aagacaaatggtgctgaagg tcncagggcg ggaaagaaaa aaaaaaaaaa 660 aa 662 28 504 DNAArtificial Sequence PCR Amplified Human 28 tttttttttt ttttttttggcttgactcag gatttaaaaa ctggaacggt gaaggtgaca 60 gcagtcggtt ggagcgagcatcccccaaag ttcacaatgt ggccgaggac tttgattgca 120 cattgttgtt tttttaatagtcattccaaa tatgagatgc gttgttacag gaagtccctt 180 gccatcctaa aagccaccccacttctctct aaggagaatg gcccagtcct ctcccaagtc 240 cacacagggg aggtgatagcattgctttcg tgtaaattat gtaatgcaaa atttttttaa 300 tcttcgcctt aatacttttttattttgttt tattttgaat gatgagcctt cgtgcccccc 360 cttccccctt ttttgtcccccaacttgaga tgtatgaagg cttttggtct ccctgggagt 420 gggtggaggc agccagggcttacctgtaca ctgacttgag accagttgaa taaaagtgca 480 cacctgaaaa aaaaaaaaaaaaaa 504 29 66 DNA Artificial Sequence Synthetic 29 tctagtcgacggccagtgaa ttgtaatacg actcactata gggcgttttt tttttttttt 60 tttttt 66

What is claimed is:
 1. A method for enrichment of natural antisensemessenger RNA (mRNA), comprising: generating a population of cDNA frompoly A+ mRNA in a sample of RNA using a reverse transcriptase enzyme anda polydeoxythymidine-containing oligonucleotide primer having at its5′-end a sequence identical to an amplification primer used in a laterstep in the method; incubating the population of generated cDNA tohybridize a sense cDNA from the population of cDNA with an antisensecDNA from the same population of cDNA, wherein the antisense cDNA has asingle-stranded segment complementary to the sense cDNA and hybridizesthereto to form a hybrid molecule with a double-stranded segment;treating the hybrid molecule with a DNA polymerase having a 5′ to 3′polymerase activity and a 3′ to 5′ exonuclease activity to remove anysingle-stranded non-hybridized segments of the hybrid molecule from 3′to 5′ and to extend the double-stranded segment of the hybrid molecule5′ to 3′ over an adjacent single-stranded segment as template, therebyforming a double-stranded molecule; amplifying the double-strandedmolecule using a thermostable polymerase and an amplification primeridentical to the sequence at the 5′-end of thepolydeoxythymidine-containing oligonucleotide primer; and cloning theamplified double-stranded molecule to enrich for a natural antisensemRNA encoded by the amplified double-stranded molecule.
 2. The methodaccording to claim 1, wherein the DNA polymerase in said treating stepis T4 DNA polymerase.
 3. The method according to claim 1, wherein theDNA polymerase in said treating step is Platinum Pfx DNA polymerase. 4.The method according to claim 1, wherein the DNA polymerase in saidtreating step is Deep Vent DNA polymerase.
 5. The method according toclaim 1, wherein the DNA polymerase in said treating step is Pwo DNApolymerase.
 6. The method according to claim 1, wherein the DNApolymerase in said treating step is Pfu DNA polymerase.
 7. The methodaccording to claim 1, wherein the polydeoxythymidine-containingoligonucleotide primer further comprises a restriction enzyme cleavagesite.
 9. The method according to claim 1, wherein the sample of RNA is amixture of RNA from two or more sources.
 10. A method for detection ofdifferential expression of natural antisense messenger RNA (mRNA),comprising: (a) separately obtaining polyA-mRNA-A molecules from cellpopulation A and polyA-mRNA-B molecules from cell population B; (b)separately generating by a reverse transcription enzyme a population ofsingle-stranded cDNA-A molecules from polyA-mRNA-A and a population ofsingle-stranded cDNA-B molecules from polyA-mRNA-B, wherein thepolydeoxythymidine containing oligonucleotide primer used to produce thecDNA-B molecules comprises a specific bacteriophage RNA polymerasepromoter region close to its 5′ terminus; (c) incubating the combinedpopulations of single-stranded cDNA-A molecules and single-strandedcDNA-B molecules, under conditions allowing hybridization of sense cDNAmolecules with antisense cDNA molecules, wherein each single-strandedantisense cDNA molecule that hybridizes has a segment complementary tothe sense DNA molecule and hybridizes thereto to form a hybrid moleculewith a double-stranded segment; (d) treating the hybrid molecules with aDNA polymerase having a 5′ to 3′ polymerase activity and a 3′ to 5′exonuclease activity to remove single-stranded non-hybridized segmentsof the hybrid molecule from 3′ to 5′ and to extend the double-strandedsegment of the hybrid molecule 5′ to 3′ over an adjacent single-strandedsegment as template, thereby forming a double-stranded molecule havingthe RNA polymerase promoter region close to one terminus; (e) using thedouble-stranded molecule as a template for the specific RNA polymeraseto produce a population of RNA molecules; (f) labeling with a firstlabel the RNA molecules produced in step (e); (g) labeling with a secondlabel as control the polyA-mRNA-A molecules and/or the polyA-mRNA-Bmolecules of step (a); (h) mixing labeled RNA molecules from steps (f)and (g) and hybridizing them to a DNA microarray; and (i) identifyingthe genes on the microarray which are preferentially labeled with thelabeled RNA molecules of step (f).
 11. The method according to claim 10,wherein the specifc bacteriophage polymerase is selected from the groupconsisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNApolymerase.
 12. The method according to claim 10, wherein following step(b) the cDNA-B is modified in order to resist the 3′ to 5′ exonucleaseactivity of step (d).
 13. The method according to claim 12, wherein instep (d) the 3′ terminus of the cDNA-B is modified.
 14. The methodaccording to claim 12, wherein in step (d) the entire cDNA-B ismodified.
 15. The method according to claim 13, wherein 3′ terminus ofthe cDNA-B is modified by addition of a nucleotide analog.
 16. Themethod according to claim 14, wherein the entire cDNA-B is modified byincorporation of nucleotide analogs.
 17. The method according to claim10, wherein the first label of step (f) is Cy3, and the second label ofstep (g) is Cy5.
 18. The method according to claim 10, wherein the firstlabel of step (f) is Cy5, and the second label of step (g) is Cy3.
 19. Amethod for detection of differential expression of natural antisensemessenger RNA (mRNA), comprising: (a) separately obtaining polyA-mRNA-Amolecules from cell population A and polyA-mRNA-B molecules from cellpopulation B; (b) separately generating by a reverse transcriptionenzyme a population of single-stranded cDNA-A molecules frompolyA-mRNA-A and a population of single-stranded cDNA-B molecules frompolyA-mRNA-B, wherein the polydeoxythymidine containing oligonucleotideprimer used to produce the cDNA-A molecules comprises close to its 5′terminus a sequence identical to an amplification primer used in step(e) and wherein the polydeoxythymidine containing oligonucleotide primerused to produce the cDNA-B molecules comprises a specific bacteriophageRNA polymerase promoter region close to its 5′ terminus; (c) incubatingthe combined populations of single-stranded cDNA-A molecules andsingle-stranded cDNA-B molecules, under conditions allowinghybridization of sense cDNA molecules with antisense cDNA molecules,wherein each single-stranded antisense cDNA molecule that hybridizes hasa segment complementary to the sense DNA molecule and hybridizes theretoto form a hybrid molecule with a double-stranded segment; (d) treatingthe hybrid molecules with a DNA polymerase having a 5′ to 3′ polymeraseactivity and a 3′ to 5′ exonuclease activity to remove single-strandednon-hybridized segments of the hybrid molecule from 3′ to 5′ and toextend the double-stranded segment of the hybrid molecule 5′ to 3′ overan adjacent single-stranded segment as template, thereby forming adouble-stranded molecule having the RNA polymerase promoter region closeto one terminus; (e) amplifying the double-stranded molecule of step (d)using a thermostable polymerase and a first amplification primeridentical to the sequence used in step (b) and a second amplificationprimer identical to the specific bacteriophage RNA polymerase promoterregion of step (b); (f) using the double-stranded molecules so producedas a template for the specific RNA polymerase to produce a population ofRNA molecules; (g) labeling with a first label the RNA moleculesproduced in step (f); (h) labeling with a second label as control thepolyA-mRNA-A molecules and/or the polyA-mRNA-B molecules of step (a);(i) mixing labeled RNA molecules from steps (g) and (h) and hybridizingthem to a DNA microarray; and (i) identifying the genes on themicroarray which are preferentially labeled with the labeled RNAmolecules of step (g).
 20. The method according to claim 19, wherein thespecifc bacteriophage polymerase is selected from the group consistingof T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.
 21. Themethod according to claim 19, wherein following step (b) the cDNA-B ismodified in order to resist the 3′ to 5′ exonuclease activity of step(d).
 22. The method according to claim 21, wherein in step (d) the 3′terminus of the cDNA-B is modified.
 23. The method according to claim21, wherein in step (d) the entire cDNA-B is modified.
 24. The methodaccording to claim 22, wherein 3′ terminus of the cDNA-B is modified byaddition of a nucleotide analog.
 25. The method according to claim 23,wherein the entire cDNA-B is modified by incorporation of nucleotideanalogs.
 26. The method according to claim 19, wherein the first labelof step (g) is Cy3, and the second label of step (h) is Cy5.
 27. Themethod according to claim 10, wherein the first label of step (g) isCy5, and the second label of step (h) is Cy3.